The loss of natural color in preserved biological specimens has been a problem to biologists for quite some time, particularly to those studying marine specimens in which a plethora of colorful specimens are available from the vast spectrum of ocean inhabitants or those interested in the actual colors of plants and other biological specimens. Taking notes, recording, and photographing specimen colors for future scientific analysis is time consuming and detracts from the more specific problems in the observation of preserved specimens. Ideally, the best situation would be to preserve the natural color of the preserved marine specimen.
In the past, various preservative media have been used with varying degrees of success for the preservation of the natural color of the marine specimen, such media being usually solutions of formaldehyde and alcohol of varying concentrations. However, the solutions are generally toxic, they have an irritating odor, and they are frequently irritating or toxic to the experimenter, and therefore, difficult to work with in the laboratory and many of these preservatives cause the preserved specimen to slowly decompose.
Formaldehyde solutions continuously age with the oxidative development of formic acid. This reaction lowers the pH and is undesirable for the preservation of calcareous materials. Also, specimen color preservation has not always been of the first quality, and large volumes of the preservative have to be stored throughout the laboratory.
In biological specimens stored in formaldehyde there is a 60% destruction of the protein materials. The protein is irreversibly denatured or precipitated like egg albumin turns white when cooked.
The commonly used preservative materials are 10% formalin solutions, 40% isopropyl alcohol solutions, and 70% ethyl alcohol solutions. Formaldehyde hardens tissues. The natural appearance of the tissues with respect to color and texture is generally quickly lost and the specimen solutions become cloudy. Specimens preserved in alcohol also lose their natural color very quickly, particularly, when exposed to light.
An object of this invention is to provide an improved biological specimen preservative in which the original color of the specimen is retained over long periods of time.
Another object ot this invention is to provide a preservative for biological specimens that is non-toxic and non-irritating to laboratory personnel.
Another object of this invention is to provide a preservative for biological specimens that may be used repeatedly by removing fixed specimens and dry packing them in plastic bags. Other objects will be apparent from the following specification.
I have discovered a new preservative composition, and a variety of preservative techniques and modifications of this composition that do not have the disadvantages of prior art formaldehyde and alcohol preservatives and dry packing biological specimen preservative techniques. With the use of these new preservative compositions continuous exposure to natural and artificial light caused no color deterioration in the particular specimens observed, the solutions do not become cloudy with age and the preserved specimens can be kept without being retained in the preservative solution, the solutions have indefinite shelf life, and the preserved specimen retains its original odor and skin texture.
The improved preserved biological specimens are obtained by using specifically modified starch glycerite solutions as the preservating agent. This modified product is a new composition.
The preservative effect appears to result in part from the dehydration of the specimen and the bacteriostatic action appears to be due to the development of a new reaction product obtained from the soluble starch glycerite in addition to a simple dehydration mechanism. Evidence that the preservatives are new compositions consists of the following:
1. Viscosity changes have been repetitively observed at about the same temperature (ca 130.degree. C. during the heat treatment step used in the preparation of the preservative).
2. These viscosity changes appear to coincide with the elimination of water vapor which indicates that a condensation reaction and formation of a new compound takes place during this heat treatment step.
3. The finished product has a ketonoid odor which is not characteristic of either of the soluble starch or glycerin reactants.
4. Specimens fixed with this preservative remain well preserved years after they are removed from the preservative solution. One sample of copepods has been stored in sea water for over one year without decay. Other specimens such as plants, small fish and animal organs have also been preserved for 1 to 4 years without undergoing any deterioration.
The starting pretreated starch glycerite solutions can be prepared in the conventional manner by dissolving a soluble starch, such as potato starch, in a solvent such as a dihydric alcohol, such as ethylene glycol, diethylene glycol, propylene glycol, etc.; trihydric alcohol, such as glycerol; or low molecular weight polypropylenes having 6 to 9 carbon atoms. The preferred starches are amylopectin starch having a molecular weight of 300-3000, amylose starch having a molecular weight of 100-1000, a mixture of these starches, or a mannose, such as monosaccharidehexoses.
Raw starch is not soluble because its molecules are too large.
To make the soluble starch, add a polyol such as glycerine, to raw starch heat the mixture to 190.degree. C., add alcohol to precipitate a product called soluble starch.
Starch glycerite is obtained by redissolving the soluble starch precipitate in glycerine by heating the mixture.
The modified starch glycerite of this invention is made by slowly redissolving the soluble starch in a polyol, such as glycerine at a redissolving-reaction temperature of about 135.degree. C. to a temperature below the carmelization temperature. The preferred reaction temperature is between 140 to 160.degree. C. Preferably the mixture is agitated during the redissolving-reaction step. This step is continued until it produces a colloidal solution which contains the preservative of this invention. This product has a ketonoid odor and a relatively large concentration of hydroxyl groups.
At 125.degree. C. the viscosity of the solution increases and it looks like syrupy milk; at 130.degree. C. the viscosity suddenly decreases significantly and a slightly turbid solution forms.
This composition is hypertonic, which means that it dehydrates biological material it comes in contact with. In cases where dehydration is undesirable, it can be reduced by dilution of the product. Since dilution may encourage the growth of bacteria, it is desirable to add a bactericide to these dilute solutions.
This colloidal solution will turn blue when subjected to an iodine indicator test which indicates that some amylodextrin is present.
An improved preservative which is a colorless solution can be made by adding about one micro liter of any mineral acid, such as phosphoric acid, to about 1000 milliliters of the colloidal product at room temperature. This acid treatment eliminates the cloudiness of the colloid solution by partially depolymerizing the carbohydrate polymers by hydrolysis and the acid acts as an antioxidant which further helps maintain the colors of biological specimens.
This clear solution does not turn blue when subjected to an iodine indicator test. Therefore the polymer product contains no starch and no amylodextrin.
The preservative solution could be further modified with one or more of the following modifiers: hydrochloric acid, sulfuric acid, sodium carbonate, acetic acid, acetic anhydride, picric acid, eugenol, menthol, any one of the monosaccharide hexoses and/or one or more of the following bacteriostats or germicides can be added to diluted solutions of the preservative; sodium benzoate, benzoic acid, sodium borate, boric acid, hexamethylenetetramine, and hydrochloric acid.
Modifiers such as those listed above, are used to alter the histochemical properties of the preservative through one or more of the following techniques:
I pH control
A. acid fixative generally non-clearing PA1 B. basic fixative generally clearing
II Molecular weight reduction through cracking or degradation of the polymer.
III Molecular weight increase via polymerization or condensation reactions.
IV Introduction of various functional groups to promote special properties in the preservative.
The composition and techniques have been effectively used to preserve phytoplankton, zooplankton, marine crustacea, fishes up to several inches in length, scallops, sea grasses, insects, reptiles, and amphibians. The starch glycerite reaction product solution may be used to preserve any biological specimen.
Specimens are preferably injected with 100% preservative into the body cavity then immersed for 24 to 48 hours, depending upon body weight, in 75% preservative. They may be stored permanently in the 75% solution or removed and packaged dry in polyethylene bags. Color retention over long periods is generally better preserved in the solution. For small and soft bodied specimens, it is more useful to pass specimens through a concentration gradient consisting of 25%, 50%, 75% and 100% of preservative. 0.1% to 2% sodium benzoate plus 1% sodium carbonate is added to each diluted solution as a bacteriostatic in the absence of formaldehyde. 4% formaldehyde may be used as the diluent in which case most of its noxious, irritant properties seem to be thoroughly suppressed by the starch glycerite reaction product.
In a preferred embodiment of the invention the modified starch glycerite colloidal solution of this invention is prepared in the following manner:
1. Add 630 or 1260 parts/weight of a polyhydric alcohol, such as glycerin to 30 to 50 parts by weight of soluble starch powder. (Preferred ratio is 630/40 parts by weight.)
2. Heat with stirring to a temperature of 135.degree. C to 190.degree. C. avoiding carmelization. The preferred temperature is 150.degree. C.
3. Optionally if 630 parts glycerine is used in step 1, when the starch powder is in solution, add an additional 630 parts by weight of glycerol and reheat to the desired temperature. Hold at this temperature for five minutes and cool to room temperature. The preservative is now ready for use.
To produce near normal turgor, the specimen may be run backwards through the concentration gradient as far as the 50% concentration. After immersion in 100% preservative for periods ranging from 24 hours to one week, depending on the particular biological specimen, the specimen may be removed from the preservative and maintained in a dry-pack container during storage. Such storage eliminates the space and cost requirement for maintaining large volumes of preservative in collections. Other advantages include the fact that preserved specimens, such as crustacea, are easier to work with (greater limberness) when preserved by this method and much greater flexibility is provided with respect to modification of the preservative for special applications. For example, the concentration may be widely varied and various additives may be used to achieve a desired effect.